Nascent RNA-seq of human AID-NeonGreen-Tpr tagged DLD-1 cells after loss of Tpr nucleoporin

We used SLAM-seq approach to define a contribution of RNA synthesis into RNA abundance changes after Tpr loss. Basket nucleoporin Tpr was AID-tagged and depleted through Auxin Induced Degradation system. Loss of Tpr led to rapid changes in rates of RNA synthesis. The majority of transcripts were downregulated. Analysis of RNA-seq and SLAM-seq indicated that the changes in RNA abundance of most of Tpr-dependent up- and downregulated genes resulted from increased or decreased rates of RNA synthesis, respectively. Overall design: Thiol (SH)-Linked Alkylation for the Metabolic (SLAM) sequencing of nascent RNAs was performed with 4-Thiouridine (S4U) incorporation using Anabolic (061.24) Kinetics Module kit purchased from Lexogen company. Briefly, before labelling of nascent RNA, each well of DLD-1 AID-Tpr cells from the 6-well plate was replaced with fresh media or media containing 1 mM auxin for 2 hours. For chasing, cells were treated with media containing 300 µM S4U or 300 µM S4U supplemented with 1 mM auxin for 0 min, 30 min, 60 min, or 2 hours. Total RNAs were processed to allow conversion of S4U to cytosine, sequenced, and the T>C reads were used to identify genes that were up- and down-regulated in response to Tpr loss.